腺相关病毒转染改良基因nd4进入线粒体

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1、华中科技大学硕士学位论文Adeno-associatedvirus-mediatedmodifiedND4geneenteringthemitochondriaDepartmentofOphthalmology,TongjiHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnologyPostgraduate:WanXingTutor:Porf.LiBinAbstractAim:ToinvestigateAAV-mediatedthemodifiedND4gen

2、ewhethercouldfacilitateND4proteinredirectimporttomitochondria.Methods:AnuclearencodedhumanND4genefusedtoCOX10mitochondrialtargetingsequence(MTS)andCOX103‘UntranslatedRegions(UTR)werepackagedinAAV2/2capsid.ThentherecombinantAAV2/2-MTS-ND4weconstructedwerevalidatedbyvitroan

3、dvivoexperiments,respectively.Invitroexperiment,cellsweredividedintothreegroupsandwereaddingPBS,AAV-GFP,rAAV-MTS-ND4insequence.ThemRNAandproteinofND4intheHumanembryonalkidneycells(293T)weredeterminedwithimmunofluorescence,Westernblotandreal-timepolymerchainreaction(RT-PCR

4、)after48hours.Invivoexperiment,animalsweredividedintothreegroupsandreceivedintravitrealinjection4μlofPBS、AAV-GFP、rAAV-MTS-ND4insequence.ThemRNAandproteinofND4inretinaofrabbitsweredeterminedwithimmunofluorescence,Westernblotandreal-timepolymerchainreaction(RT-PCR)atthirtie

5、thdayspost-injection.Results:ImmunofluorescenceofcellsshowND4proteinwereredirectimportedtomitochondriaofcells.IntravitrealinjectionofAAVvectorsefficientlytransducedmainlyretinalganglioncells.WesternblotanalysesverifiedthesignificantlyincreasedexpressionsofND4proteinintomi

6、tochondrialof293cellsandretinalganglioncellsofmouseingroup3thantheothertwogroups.Real-timepolymerchainreaction(RT-PCR)detectionverifiedthesignificantlyincreasedmRNAofND4in293cellsandretinalganglioncellsofmouseingroup3thantheothertwogroups.Conclusions:TherecombinantAAV2/2-

7、MTS-ND4weconstructedcouldmakeND4proteinredirectimportedtomitochondriaofretinalganglioncellsofrabbits,then2万方数据华中科技大学硕士学位论文layingagoodfoundationforclinicaltreatmentofLHONinfuture.Keywords：LHON;genetherapy;mitochondrialtargetingsequence(MTS);intravitrealinjection3万方数据华中科技大学

8、硕士学位论文英文缩略语缩略语英文全称中文全称LHONLeber‘shereditaryopticneuropathyLeber遗传性视神经病变MTSMitochondrialtargeting